Abstract:

Objective: To investigate the in vitro proliferation and osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) of SD rats in different generations. Methods: Primary BMSCs were isolated and cultured from rats, and BMSCs were identified using immunofluorescence analysis. The BMSCs were subcultured to obtain P1, P3, P5, P7 and P9 generations of BMSCs. The proliferation of BMSCs in different generations were tested by CCK-8 assay. The cell morphology of BMSCs in different generations were analyzed under microscope and immunofluorescence assay was used for cytoskeleton protein evaluation. Flow cytometry was applied to identify cell apoptosis. Both alkaline phosphatase (ALP) and alizarin red staining tested the osteogenic differentiation ability of BMSCs in different generations in vitro. Real-time polymerase chain reaction (RT-PCR) and immunofluorescence were used to detect the gene expression and protein level of osteogenic marker collagen type Ⅰ α1(COL1A1) and osteocalcin (OCN). Results: There were no significant differences in the morphology, proliferation, cell apoptosis and osteogenic differentiation between P1, P3, P5 generations of BMSCs, but remarkable decrease was observed in P7 and P9 generations of BMSCs more than P1 generation. Conclusion: After 5 generations, the proliferation and osteogenic differentiation ability of BMSCs significantly decreased with aging.

Key words: different generations, bone marrow mesenchymal stem cells, osteogenic differentiation, tissue engineering, cytoskeleton

摘要：

目的: 探讨不同代数SD大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）体外增殖和成骨分化能力的差异。方法: 从SD大鼠骨髓中分离并培养原代BMSCs，免疫荧光染色鉴定BMSCs。传代培养分别获取P1、P3、P5、P7、P9代BMSCs。CCK-8实验检测不同代数BMSCs的增殖能力；显微镜下观察细胞形态学特征；免疫荧光染色检测细胞骨架；流式细胞术检测细胞凋亡情况；碱性磷酸酶（alkaline phosphatase，ALP）染色和茜素红染色比较不同代数BMSCs的体外成骨分化能力；实时聚合酶链式反应（real-time polymerase chain reaction，RT-PCR）和免疫荧光检测不同代数BMSCs成骨分化标志物Ⅰ型胶原A1（collagen type Ⅰ α1，COL1A1）、骨钙素（osteocalcin，OCN）基因表达和蛋白水平。结果: P1、P3、P5代间BMSCs的细胞形态、体外增殖、细胞凋亡及成骨分化能力无显著差别；而P7和P9代BMSCs各方面指标与P1代BMSCs比较，均明显降低（P<0.05）。结论: 5代以后的BMSCs随着传代次数的增加，其细胞增殖和成骨分化能力显著降低。

关键词: 不同代数, 骨髓间充质干细胞, 成骨分化, 组织工程, 细胞骨架

CLC Number: 

• R782